The mammalian liver beta-glucuronidase (E.C.3.2.1.31) catalyzed hydrolysis of 6-mercaptopurine S-beta-D-glucuronide to the anticancer drug 6-mercaptopurine and glucuronic acid is characterized by a burst of 6-mercaptopurine followed by a steady state release of this compound, indicating the formation of glucuronosyl glucuronidase on the reaction path. It is proposed to: 1. establish the SN2-general acid catalysis mechanism for beta-glucuronidase and to identify catalytic residues involved; 2. study effects of substituents in p-substituted phenyl S-beta-D-glucuronides on rate constants for glycosyl enzyme formation with a view toward understanding the nature of the transition state for reaction; 3. synthesize and test prototype reversible and irreversible beta-glucuronidase inhibitors; 4. test for the selective accumulation of 6-mercaptopurine in tumor tissue (vs. normal muscle tissue) of test rats fed glucose prior to, and during dosing with 6-mercaptopurine beta-D-glucuronide. These objectives shall be approached by use of presteady state and steady state kinetics methods which shall involve stopped-flow and conventional spectrophotometry and use of group-specific protein reagents: the combined kinetics and chemical approach is intended to reveal quantitatively mechanisms and identify catalytic amino acid participants. Design and synthesis of active-site-directed irreversible inhibitors and reaction of beta-glucuronidase with them should contribute to the above goal as well as provide information useful in designing an isoenzyme-specific inhibitor of beta-glucuronidase of potential therapeutic utility. Testing for the selective accumulation of 6-mercaptopurine in tum r tissue shall be accomplished by extraction of animal tissue and testing quantitatively via chromatography and spectrophotometry techniques for 6-mercaptopurine.